Hepatitis C virus (HCV) infection is the most important cause of chronic liver disease in the US and a rapidly growing cause of hepatocellular carcinoma (HCC) worldwide. Our understanding of the cellular factors that regulate HCV replication has been limited by the paucity of tissue culture models supportive of viral replication. Two parallel developments now permit steps to be taken to comprehensively screen for cellular proteins that participate in the viral lifecycle. The first has been development of cell-based models of HCV replication, including one developed in our laboratory that is capable of sustained HCV (+) and (-) strand as well as protein synthesis. We have adapted an assay for HCV core protein production as a simple readout for this model. The second development has been the emergence of high throughput screens of large chemical libraries that permit identification of host proteins that regulate biological processes. We therefore propose to take steps toward development of a high-throughput chemical genetic screen to identify cellular regulators of HCV replication using our cell-based assay as a phenotypic readout. Preliminary data indicate that we can successfully convert our cell-based replication assay to the more tractable 96-well format and that replication is reproducibly regulated by interferon and dexamethasone. During this proposal period, we will (1) optimize miniaturization of our cell-based assay to the 96-well format; and (2) begin screening of chemical libraries in collaboration with the Institute for Chemistry and Cell Biology at Harvard Medical School using known biologically active compounds. Initial hits identified from primary screens will be confirmed using secondary biological assays, including real-time assays for HCV RNA. Ultimately, affinity approaches using small molecule "hits" as probes will be planned to identify cellular proteins that regulate HCV replication. The use of this assay system to screen large chemical libraries will have two important consequences in studies that emanate from the proposed planning study. First, it will serve as an antiviral discovery screen. Initial hits will then be analyzed by secondary and tertiary screens to discern structure-activity relationships. Second, discovery of hit compounds that positively or negatively regulate HCV replication will ultimately help lead to identification of novel cellular targets that can be exploited to further interrupt the viral lifecycle. [unreadable] [unreadable]